A liver has 500 or more types of various specific functions. Major functions of a liver include plasma protein synthesis and secretion, blood sugar control by gluconeogenesis and glycogen metabolism, lipogenesis, ureogenesis, bile synthesis and secretion and detoxication.
Most substances incorporated into a body are mainly metabolized in a liver. In the filed of pharmaceutical development, what type of metabolism pharmaceutical candidate substances will be received and what type of effect is given to a liver or other organs and tissues are essential data. Further, many chemical substances have been synthesized and discharged into environment up to now. To elucidate what kind of effect these substances have exerted individually or in combination to a human body is socially very important. Toxicological tests on liver function are essential for evaluation of the effects of such chemical substances to a human body.
Mice, rats, rabbits, dogs, monkeys, etc are used at present for safety tests and drug metabolism tests of chemical substances including pharmaceutical candidate substances. Especially in pharmaceutical development, toxicological tests and safety tests using animals are compulsive before entering phase I study for human, and long period and efforts as well as huge costs are required in these tests.
However, there is no guarantee that data obtained by these animal experiments can be applicable to human. In fact, many cases are known wherein substances not recognized toxicity in animal experiments exhibited toxicity in human or vice versa. Consequently, there might exist many cases wherein development of many pharmaceutical candidate substances were terminated after entering in phase I study on human, or also many substances were terminated development before entering clinical trials due to exhibiting strong toxicity in animal experiments but recognized actually no toxicity in human.
This may be caused by difference in metabolic function in a human liver and in metabolic function in livers of mice and rats. Recently, in vitro metabolic tests and toxicity tests using human hepatocytes have been performed. However, amount of livers from brain death patients which were not used for transplantation and amount of human hepatocytes obtained from hepatectomy in tumor excision are far fewer than demanded. Consequently, development of technology for human hepatocyte proliferation is essential for pharmaceutical development.
Necessity of high amount of human hepatocytes is very much alike in an extracorporeal artificial liver. The artificial liver is medical device acting liver function artificially. Development of a hybrid artificial liver combining with the artificial function based on physico-chemical principle such as adsorption, dialysis and filtration, along with biological actions using perfusion of an excised liver and liver tissue is strongly progressing. In development of the artificial liver, performance improvement of membrane and circuit to enhance physico-chemical function is essential, along with supplying high amount of hepatocytes applicable to human use.
However, subcultivation of primary cultured cells of a human liver isolated from adults has been considered impossible. Namely, the matured hepatocytes with adhesion dependency are largely damaged when cells are detached from culture substrate for subcultivation operation and are difficult to re-adhere to culture substrate. Contrary to that, the present inventors have invented a method for proliferative hepatocytes comprising isolating small hepatocytes, having clonal proliferative ability, from normal hepatocytes isolated from human liver, performing primary culture of the small hepatocytes, and subculturing further the cultured hepatocytes, and the invention was granted patents (JP-A-08-112092; JP No. 3266766; U.S. Pat. No. 6,004,810, JP-A-10-179148; JP No. 3211941, JP-A-07-274951; JP No. 3157984, and JP-A-09-313172; JP No. 3014322). Relating articles published are as follows: (Tateno, C. and Yoshizato, K.; “Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells”, Am. J. Phathol. 149: 1593–1605, 1996; Hino, H. Tateno, C. Sato, H. Yamasaki, C. Katayama, S. Kohashi, T. Aratani, A. Asahara, T. Dohi, K. and Yoshizato, K.; “A long-term culture of human hepatocytes which show a high growth potential and express their differentiated phenotypes”, Biochem. and Biophys. Res. Commun. 256: 184–191, 1999; Tateno, C. Kajihara, K. T. Yamasaki, C. Sato, H. and Yoshizato, K. “Heterogeneity of growth potential of adult rat hepatocytes in vitro”, Hepatology 31: 65–74, 2000; and Katayama, S. Tateno, C. Asahara, T. and Yoshizato, K.; “Size-dependent in vivo growth potential of adult rat hepatocytes”, Am. J. Pathol. 158: 97–105, 2001).
The method of the above prior patented inventions provides novel means for obtaining high amount of human hepatocytes by proliferative hepatocytes in vitro, however, the human hepatocytes obtained by this method have a problem that various liver functions are impaired during long-term subculture. There is further a problem that the small hepatocytes can not differentiate to hepatocytes (functional hepatocytes) having function equivalent to normal hepatocytes, for example, albumin expression level and metabolic activity of cytochrome P450 for chemical substance and so on. By those reasons, they are still insufficient as hepatocytes for alternative human liver function or as a material for a hybrid artificial liver, although they are useful as a screening system of drugs for maintaining specific liver function or as a system for testing toxicity and pharmacological effect of drugs on specific functions which are conserved after long-term subculture.
Small hepatocytes having proliferation potency can be collected by using not only a method for centrifugal separation as described in the above prior patented invention (a method for separating cells in supernatant obtained by low speed centrifugation) but also a cell sorter such as an elutriator and FACS, however, cells obtained by such means are a mixture of not only proliferative hepatocytes but also other cells (such as non-hepatic parenchymal cells such as stellate cells contained in supernatant by low speed centrifugation). Consequently, means to obtain substantially proliferative human hepatocytes only has been required.
The present invention has been completed considering the above circumstances, and an aspect of the present invention is to provide a monoclonal antibody which specifically recognizes proliferative human hepatocytes.
Further, other aspect of the present invention is to provide a method for isolating proliferative human hepatocytes and the proliferative human hepatocytes obtained by such a method.
Further, other aspect of the present invention is to provide a method for inducing to differentiate the proliferative human hepatocytes to functional human hepatocytes, the functional human hepatocytes obtained by such a method and utilization of the functional human hepatocytes.